To study how G protein-coupled receptors (GPCRs) are assembled and function at a molecular level, various members of the muscarinic acetylcholine receptor family (m1-m5) were used as model systems. Coexpression studies with m3 muscarinic receptor fragments (obtained by splitting the receptor in all three intracellular (i1-i3) and all three extracellular loops) indicated that GPCRs are assembled from multiple autonomous folding domains. This finding was also confirmed by immunocytochemical studies showing that most receptor fragments examined were stably incorporated into lipid bilayers (including the plasma membrane), even when expressed alone. Hybrid m2/m5 muscaninic receptors that contain m5 sequence in transmembrane domain I (TM I) and m2 sequence in TM VII are known to be pharmacologically inactive. We could demonstrate that such misfolded hybrid receptors can be pharmacologically rescued by introduction of a single point mutation into either TM I (m5Thr37->m2Ala3O) or TM VII (m2Thr423->m5His478). This finding allows predictions as to how TM I and TM VII are oriented relative to each other. To identify single amino acids required for selective coupling of the m3 receptor to G proteins of the (Gq/11 family, distinct intracellular segments/amino acids of the m3 receptor were systematically substituted into the Gi/o-coupled m2 receptor. Functional analysis of the resulting mutant receptors showed that selective recognition of Gq/11 by the m3 receptor is determined by a limited number of amino acids located in the i2 loop and at theN- and C-terminus of the i3 domain. To delineate specific sites of contact between the m2 receptor and Gi/o proteins, coexpression studies were carried out with mutant m2 receptors and G protein alpha-q-subunits in which the C-terminal portion was replaced with sequences derived from alpha-i2 or alpha-o. These studies showed that a short region in the m2 receptor, located at the junction between the i3 loop and TM VI, can recognize the C-terminus of G-alpha-i (G-alpha-o) subunits with high selectivity. Since all GPCRs share a similar molecular architecture, our findings should be of broad general relevance.